Despite the importance of platelets in normal physiology and disease, the mechanism by which they are formed within the megakaryocyte cytoplasm is poorly understood. The formation of proplatelets and ultimately platelets requires several processes, including remodeling of the actin cytoskeleton, assembly of the marginal band of microtubules, and incorporation of the multiple receptors that are required for platelet activation and adhesion into the plasma membrane. The goal of this project is to investigate the molecular mechanism of platelet production using a model system in which platelets are formed in vitro from mouse megakaryocytes. Preliminary characterization of "platelets" from this cell culture system revealed they are ultrastructurally similar to blood platelets and video-enhanced real-time light microscopy of in vitro platelet formation has identified key aspects of this process. The specific aims of this proposal are to determine if platelets released in culture are functionally equivalent to those isolated from blood, determine the ultrastructure and dynamics of the cytoskeleton during specific stages of platelet production using established microscopy techniques, and determine when key platelet proteins are produced by establishing the biochemical composition of megakaryocyte cytoplasm at specific stages of platelet formation.